Many molecular biological methods require a cloning step which requires the use of a DNA or RNA ligase to attach oligonucleotide adaptors or other sequences to a target nucleotide sequence. The efficiency of this ligation is dependent on the sequence of the adaptor and the target. When creating a library of sequences from RNA or DNA it is often important to ligate all possible sequences and also for the library to be representative of the relative abundance of the target. These two properties are important when generating high quality libraries of small RNAs. These libraries can be sequenced using traditional Sanger sequencing but are now more commonly sequenced by high throughput sequencing or Next Generation Sequencing (NGS) techniques.
Eukaryotic gene expression is regulated at several layers and one of the most recently discovered mechanisms involves small, 20-24 nucleotide (nt) long, non-coding RNA molecules (sRNAs) (Fire, Xu et al. 1998; Voinnet 2002). There are several classes of sRNAs with different biogenesis pathways and modes of action. The best characterised class are the microRNAs (miRNAs) that are generated from stem-loop structures and target mRNAs in trans. Most miRNAs are expressed specifically in certain tissues and at specific developmental stages and their accumulation often changes due to external cues and during disease (MicroRNAs in Cancer Translational Research, William C. S. Cho, 2011, Springer). Several databases have compiled the association of miRNAs and disease including the human microRNA disease database and mir2disease (www.mir2disease.org/; 202.38.126.151/hmdd/mirna/md/) and new products have recently been launched to classify cancers (or diseases) based on their miRNA profiles, such as Mirview (Rosetta Genomics, US 2010/0273172).
Therefore, accurately profiling the level of miRNAs (and other classes of sRNAs) is very important in both basic and clinical research. Identifying an interesting miRNA for further study is an empirical process that is often based on its high expression and clear differential expression. These criteria are more important when the biological context of the miRNA is lacking such as in animals where target prediction is poor (Dalmay 2008). In addition, expression level is often used to discern between the miRNA and miRNA star. Accurate miRNA profiling is complicated by the heterogeneous nature of miRNAs such as sequence isoforms and length isomirs since these are thought to have differential activities (Fernandez-Valverde, Taft et al. 2010; Guo and Lu 2010; Starega-Roslan, Krol et al. 2011).
Measuring microRNAs with sequencing technologies such as high throughput and Sanger or by quantitative PCR (QPCR) requires the use of nucleic acid modifying enzymes. Ligases, reverse transcriptases and DNA polymerases are some of the most important enzymes used in molecular biology. To improve the activity of these enzymes their function needs to be fully understood, which requires a method of measuring their activity and identifying the determinants that regulate their function.